Methods and compositions for preventing or treating tissue calcification

ABSTRACT

The invention provides methods and compositions for preventing or treating (e.g., slowing the progression of, arresting, and/or reversing) tissue calcification in a subject in need thereof and, more particularly, the invention relates to methods of using menaquinone-7 (MK-7) and/or menaquinol-7 (MKH2-7) for preventing or treating (e.g., slowing the progression of, arresting, and/or reversing) tissue calcification in a subject with diabetes, chronic kidney disease, end stage renal failure, or a subject undergoing hemodialysis and/or receiving anticoagulant therapy. The invention further provides methods and compositions for reducing one or more symptoms of chronic obstructive pulmonary disorder (COPD), including using menaquinone-7 (MK-7) and/or menaquinol-7 (MKH2-7), for preventing or treating (e.g., slowing the progression of, arresting, and/or reversing) one or more symptoms of COPD.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalPatent Application No. 62/682,796, filed on Jun. 8, 2018; which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates generally to methods and compositions forpreventing or treating (e.g., slowing the progression of, arresting,and/or reversing) tissue calcification in a subject in need thereof and,more particularly, the invention relates to methods of usingmenaquinone-7 (MK-7) and/or menaquinol-7 (MKH2-7), for preventing ortreating (e.g., slowing the progression of, arresting, and/or reversing)tissue calcification in a subject with diabetes, chronic kidney disease(CKD), end stage renal disease, or a subject undergoing hemodialysisand/or receiving anticoagulant therapy and/or statin therapy. Theinvention further relates to methods and compositions for reducing oneor more symptoms of chronic obstructive pulmonary disorder (COPD),including using menaquinone-7 (MK-7) and/or menaquinol-7 (MKH2-7), forpreventing or treating (e.g., slowing the progression of, arresting,and/or reversing) one or more symptoms of COPD.

BACKGROUND

Under normal physiological conditions, plasma calcium and phosphate arepresent at concentrations close to supersaturation levels and, as aresult, may be expected to precipitate in soft tissue (e.g., bloodvessels) as crystalline hydroxyapatite. The observation that thisprocess does not occur in healthy subjects suggested the presence ofpotent chemical and biologic means for blocking pathologic calcification(Price, et al. (2002) “Discovery of a High Molecular Weight Complex ofCalcium, Phosphate, Fetuin, and Matrix-Carboxyglutamic Acid Protein inthe Serum of Etidronate-treated Rats,” JOURNAL BIOL. CHEM. 277 (6):3926-3934).

When the suppression of calcification is disrupted, such as in subjectswith diabetes and chronic kidney disease (CKD), pathologic calcificationof soft tissue (e.g., blood vessels) can occur. It is understood thatdiabetes can lead to CKD and end stage renal disease (ESRD), which ischaracterized by uremia. Uremia can promote the oxidation of Vitamin Khydroquinone (KH2), thereby disrupting the cyclic regeneration ofVitamin K, among other effects. (See, FIG. 1.) In addition, certaintreatments can cause or contribute to Vitamin K dysregulation, includingwarfarin-based anticoagulant therapy and statin therapy. The loss offunctional Vitamin K results in the loss of important regulators ofmineralization, leading to pathologic calcification of tissue. In thecase of arterial calcification, intradermal microvascular thrombosis isobserved to occur, resulting in small vessel blockages and surroundingtissue death.

Vitamin K is an essential enzymatic co-factor that is required forpost-translational modifications of Vitamin K-dependent (VKD) proteins.A number of VKD proteins are clinically relevant to CKD and ESRDpatients, and include, for example, central coagulation factors such asfactors II, VII, IX, and X and intercellular matrix proteins such asMatrix Gla Protein (MGP) activated protein C and osteocalcin. Vitamin Kis a group of fat soluble vitamins, which include, among other things,Vitamin K₁ (also known as phylloquinone), which is made by plants, andVitamin K₂ (also known as menaquinone), which is made by bacteria in gutflora. It is understood that the isoprenoid chain in Vitamin K₂ cancontain from 4 to 12 repeating isoprenoid units. For example,menaquinone-4 (or MK-4) contains four isoprenoid units whereasmenaquinone-7 (or MK-7) contains seven isoprenoid units.

With regard to menaquinone-7 (MK-7), under normal conditions MK-7 isreduced to menaquinol-7 (MKH2-7) (a form of Vitamin K hydroquinone) byan NADPH-dependent reductase enzyme or enzymes (e.g., quinoneoxidoreductase). Only the reduced form of MK-7 (namely MKH2-7) functionsas a co-factor for the enzyme gamma glutamate carboxylase (GGCX), whichcatalyzes the carboxylation of Vitamin K-dependent proteins. (See, FIGS.1 and 2.) The enzymatic carboxylation of glutamate residues results inoxidation of MKH2-7 to a 2,3-epoxide form (MK-7 2,3-epoxide). The finalstep of the Vitamin K cycle requires the enzymatic reduction of VitaminMK-7 2,3-epoxide back to MK-7 by Vitamin K epoxide reductase complexsubunit 1 (VKORC1, also referred to as VKOR. In some tissues, theparalog VKORC1L1 (VKORC1-Like-1) may also perform this catalyticreaction. It is believed that warfarin blocks both the generation ofMKH2-7, the active form of Vitamin K₂, as well as the regeneration ofMK-7 from Vitamin MK-7 2,3-epoxide, which may lead to the higherincidence of calcification seen among patients receiving warfarintherapy.

Despite efforts to date, there is a need for new clinical approaches toprevent and/or reverse pathologic calcification. In particular, there isa need for new clinical approaches to prevent and/or reverse pathologiccalcification in subjects with diabetes, CKD, ESRD, and subjectsreceiving anticoagulant and/or statin therapy.

SUMMARY OF THE INVENTION

It has been discovered that menaquinone-7 (MK-7) and/or menaquinol-7(MKH2-7), can be used effectively at high doses, e.g., in doses of atleast 2 mg per day, to prevent, slow the progression of, arrest, and/orreverse tissue calcification in a subject, e.g., a subject with diabetesand/or chronic kidney disease.

In one aspect, the invention provides a method of preventing or treating(e.g., slowing the progression of, arresting, and/or reversing) tissuecalcification in a subject with diabetes, chronic kidney disease or acombination thereof, and in need thereof, the method comprisingadministering to the subject at least 2 mg of substantially pure MK-7,MKH2-7, or a combination thereof, per day, thereby to prevent or treat(e.g., slow the progression of, arrest, and/or reverse) tissuecalcification, wherein the MK-7, MKH2-7 or the combination thereof isadministered in the form of a pharmaceutical composition. In certainembodiments, the subject is undergoing hemodialysis. In certainembodiments, the pharmaceutical composition comprises MK-7. In certainembodiments, the pharmaceutical composition comprises MKH2-7. In certainembodiments, the pharmaceutical composition comprises a combination ofMK-7 and MKH2-7.

In another aspect, the invention provides a method of preventing ortreating (e.g., slowing the progression of, arresting, and/or reversing)tissue calcification in a subject undergoing hemodialysis, and in needthereof, the method comprising administering to the subject at least 2mg of substantially pure MK-7, MKH2-7 or a combination thereof, per day,thereby to prevent or treat (e.g., slow the progression of, arrest,and/or reverse) tissue calcification, wherein the MK-7, MKH2-7 or thecombination thereof, is administered in the form of a pharmaceuticalcomposition. In certain embodiments, the pharmaceutical compositioncomprises MK-7. In certain embodiments, the pharmaceutical compositioncomprises MKH2-7. In certain embodiments, the pharmaceutical compositioncomprises a combination of MK-7 and MKH2-7.

In certain embodiments of any of the above aspects, the subject hasdiabetes, e.g., type II diabetes. In certain embodiments, the subjecthas chronic kidney disease, e.g., stage 1, stage 2, stage 3, or endstage renal disease (ESRD), e.g., stage 4 or stage 5. In certainembodiments, the subject is receiving non-warfarin-based anticoagulanttherapy, such as an oral anti-coagulation therapy.

In another aspect, the invention provides a method of preventing ortreating (e.g., slowing the progression of, arresting, and/or reversing)tissue calcification in a subject with stage 5 chronic kidney diseaseand undergoing simultaneous oral, non-warfarin-based anticoagulanttherapy, and in need thereof. The method comprises administering to thesubject at least 2 mg of substantially pure MK-7, MKH2-7, or acombination thereof, per day, thereby to prevent or treat (e.g., slowthe progression of, arrest, and/or reverse) tissue calcification in thesubject, wherein the MK-7, MKH2-7 or the combination thereof isadministered in the form of a pharmaceutical composition. In certainembodiments, the pharmaceutical composition comprises MK-7. In certainembodiments, the pharmaceutical composition comprises MKH2-7. In certainembodiments, the pharmaceutical composition comprises a combination ofMK-7 and MKH2-7. In certain embodiments, the subject is diabetic. Incertain embodiments, the subject is undergoing hemodialysis.

In certain embodiments of any of the above aspects, administration ofthe MK-7 and/or MKH2-7 to the subject increases the subject's serum T50value (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% or more) relativeto the subject's serum T50 value prior to administration of therespective MK-7 and/or MKH2-7.

In certain embodiments of any of the above aspects, administration ofthe MK-7 and/or MKH2-7 increases a ratio of a carboxylated to anon-carboxylated Vitamin K-dependent protein in the subject's plasma(e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or more) relative tothe ratio prior to administration of the respective MK-7 and/or MKH2-7.In certain embodiments of any of the above aspects, administration ofthe MK-7 and/or MKH2-7 decreases an amount of a non-carboxylated VitaminK-dependent protein in the subject's plasma (e.g., by at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100%, or more) relative to the amount prior toadministration of the respective MK-7 and/or MKH2-7. In certainembodiments, the Vitamin K-dependent protein is selected from Matrix GlaProtein (MGP), Growth Arrest Specific Gene 6 (Gas-6) protein, PIVKA-IIprotein, osteocalcin, activated Protein C, activated Protein S, factorII, factor VII, factor IX, and factor X.

In certain embodiments of any of the above aspects, administration ofthe MK-7 and/or MKH2-7 increases the plasma level of osteoprotegerin orFetuin A (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or more)relative to the plasma concentration of osteoprotegerin or Fetuin Aprior to administration of the respective MK-7 and/or MKH2-7.

In certain embodiments of any of the above aspects, administration ofthe MK-7 and/or MKH2-7 decreases the plasma level of D-Dimer or HighlySensitive C Reactive Protein (hs-CRP) (e.g., by at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100%, or more) relative to the plasma concentration ofD-Dimer or Highly Sensitive C Reactive Protein (hs-CRP) prior toadministration of the respective MK-7 and/or MKH2-7.

In certain embodiments of any of the above aspects, the subject has adermal and/or vascular lesion, and the administration of the MK-7 and/orMKH2-7 reduces the size of the dermal and/or vascular lesion compared tothe size of the lesion prior onset of the treatment regimen. In certainembodiments, administration of the MK-7 and/or MKH2-7 reduces the totalsurface area of the lesion by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

In yet another aspect, the invention provides a method of preventing ortreating (e.g., slowing the progression of, arresting, and/or reversing)tissue calcification in a subject in need thereof, the method comprisingadministering to the subject at least 2 mg of substantially puremenaquinone-7 (MK-7), menaquinol-7 (MKH2-7), or a combination thereof,per day so as to cause at least one, or a combination, of the following:(i) increase the subject's serum T50 value (e.g., by at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% or more) relative to the subject's serum T50value prior to administration of the MK-7 and/or MKH2-7, or (ii)increase a ratio of a carboxylated to a non-carboxylated form of aVitamin K-dependent protein in the subject's plasma (e.g., by at least5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100% or more) relative to the ratio prior toadministration of the MK-7 and/or MKH2-7, (iv) increase the plasma levelof osteoprotegerin or Fetuin A (e.g., by at least 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 100% or more) relative to the plasma concentration ofosteoprotegerin or Fetuin A prior to administration of the MK-7 and/orMKH2-7, or (v) decrease the plasma level of D-Dimer or Highly SensitiveC Reactive Protein (hs-CRP) (e.g., by at least 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%) relative to the plasma concentration of D-Dimer or HighlySensitive C Reactive Protein (hs-CRP) prior to administration of theMK-7 and/or MKH2-7, thereby to prevent or treat (e.g., slow theprogression of, arrest, and/or reverse) tissue calcification in thesubject, wherein the MK-7, MKH2-7 or the combination thereof isadministered in the form of a pharmaceutical composition. In certainembodiments, the pharmaceutical composition comprises MK-7. In certainembodiments, the pharmaceutical composition comprises MKH2-7. In certainembodiments, the pharmaceutical composition comprises a combination ofMK-7 and MKH2-7. The Vitamin K-dependent protein can be selected fromMatrix Gla Protein, Growth Arrest Specific Gene 6 (Gas-6) protein,PIVKA-II protein, osteocalcin, activated Protein C, activated Protein S,factor II, factor VII, factor IX, and factor X.

In another aspect, the invention provides a method of preventing ortreating (e.g., slowing the progression of, arresting, and/or reversing)tissue calcification in a subject in need thereof, the method comprisingadministering to the subject at least 2 mg of substantially puremenaquinone-7 (MK-7) and/or menaquinol-7 (MKH2-7) per day and a statin.In certain embodiments, the statin is selected from simvastatin,lovastatin, atorvastatin, pravastatin, pitavastatin, rosuvastatin, andfluvastatin. In certain embodiments, the MK-7 and/or MKH2-7 isadministered in the same dosage form as the statin. In otherembodiments, the MK-7 and/or MKH2-7 is administered in a separate dosageform from the statin. In certain embodiments, administration of the MK-7and/or MKH2-7 prevents or decreases the Vitamin K-depleting effects ofthe statin.

In another aspect, the invention provides a method of improving aorticcompliance in a subject in need thereof, the method comprisingadministering to the subject an effective amount of substantially puremenaquinone-7 (MK-7) and/or menaquinol-7 (MKH2-7) per day.

In another aspect, the invention provides a method of preventing ortreating (e.g., slowing the progression of, arresting, and/or reversing)peripheral vasculopathy in a subject in need thereof, wherein thesubject has ESRD or CKD, the method comprising administering to thesubject an effective amount of substantially pure menaquinone-7 (MK-7)and/or menaquinol-7 (MKH2-7) per day.

In another aspect, invention provides a method of preventing or treating(e.g., slowing the progression of, arresting, and/or reversing) one ormore symptoms of chronic obstructive pulmonary disease (COPD) in asubject in need thereof, the method comprising administering to thesubject at least 2 mg of substantially pure menaquinone-7 (MK-7) and/ormenaquinol-7 (MKH2-7) per day, thereby to prevent or treat (e.g., slowthe progression of, arrest, or reverse) the one or more symptoms ofCOPD, wherein the MK7 and/or MKH2-7 is administered in a pharmaceuticalcomposition. In certain embodiments, the one or more symptoms isselected from the group consisting of breathing difficulty, cough, mucusproduction, wheezing, and elastinolysis.

In certain embodiments of any of the above aspects, the subject hasdiabetes, e.g., type II diabetes or has been diagnosed as pre-diabetic.In certain embodiments, the subject has chronic kidney disease, e.g.,stage 1, stage 2, stage 3 or end stage renal disease (ESRD), e.g., stage4, or stage 5. In certain embodiments, the subject is receivingnon-warfarin-based anticoagulant therapy, such as an oralanti-coagulation therapy.

In certain embodiments, if non-warfarin-based anti-coagulation therapyis used, the anti-coagulation therapy can comprise an inhibitor ofFactor Xa activity (e.g., apixaban, rivaroxaban, betrixaban, edoxaban,or fondaparinux) or Factor IIa activity (e.g., dabigratran orargatroban). In certain embodiments, the subject has previously beenexposed to warfarin-based anti-coagulation therapy.

In certain embodiments, the subject is receiving a statin. For example,the statin can be selected from simvastatin, lovastatin, atorvastatin,pravastatin, pitavastatin, rosuvastatin, and fluvastatin.

In certain embodiments, the tissue calcification is a soft tissuecalcification. The tissue calcification can be, for example, a vascularor dermal calcification.

In certain embodiments of any of the above aspects, the method caninclude administering from about 2 mg to about 1,000 mg of MK-7 and/orMKH2-7 to the subject per day. In other embodiments, the method caninclude administering from about 5 mg to about 1,000 mg of MK-7 and/orMKH2-7 to the subject per day.

In certain embodiments of any of the above aspects, the method caninclude administering from about 2 mg to about 750 mg of MK-7 and/orMKH2-7 to the subject per day. In other embodiments, the method caninclude administering from about 5 mg to about 750 mg of MK-7 and/orMKH2-7 to the subject per day. In certain embodiments of any of theabove aspects, the method can include administering from about 2 mg toabout 500 mg of MK-7 and/or MKH2-7 to the subject per day. In otherembodiments, the method can include administering from about 5 mg toabout 500 mg of MK-7 and/or MKH2-7 to the subject per day. In certainembodiments of any of the above aspects, the method can includeadministering from about 2 mg to about 250 mg of MK-7 and/or MKH2-7 tothe subject per day. In other embodiments, the method can includeadministering from about 5 mg to about 250 mg of MK-7 and/or MKH2-7 tothe subject per day. In certain embodiments of any of the above aspects,the method can include administering from about 2 mg to about 100 mg ofMK-7 and/or MKH2-7 to the subject per day. In other embodiments, themethod can include administering from about 5 mg to about 100 mg of MK-7and/or MKH2-7 to the subject per day. In other embodiments, the methodcan include administering from about 10 mg to about 75 mg of MK-7 and/orMKH2-7 to the subject per day, e.g., administering 10, 25, 50 or 75 mgof MK-7 and/or MKH2-7 to the subject per day.

In certain embodiments, the MK-7 and/or MKH2-7 is administered to thesubject for at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 6months, 1 year, or indefinitely. If the subject is undergoinghemodialysis, the MK-7 and/or MKH2-7 can be administered to the subjectfor a period that includes at least the duration of hemodialysis.

In certain embodiments, the MK-7 and/or MKH2-7 is administered orally.The MK-7 and/or MKH2-7 can be disposed within a tablet, caplet orcapsule.

The description above describes multiple aspects and embodiments of theinvention. The patent application specifically contemplates allcombinations and permutations of the aspects and embodiments. These andother aspects and features of the invention are described in thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of theinvention will become apparent from the following description ofpreferred embodiments, as illustrated in the accompanying drawings. Likereferenced elements identify common features in the correspondingdrawings, in which:

FIG. 1 is a schematic diagram depicting the Vitamin K cycle and theeffects of uremia and hemodialysis-induced oxidation of Vitamin Khydroquinone; and

FIG. 2 is a schematic diagram depicting the carboxylation of VitaminK-dependent proteins by gamma-glutamate carboxylase (GGCX).

DETAILED DESCRIPTION

The invention is based, in part, upon the discovery that menaquinone-7(MK-7) and/or menaquinol-7 (MKH2-7), the reduced form of MK-7, can beadministered to a subject in need thereof to prevent or treat (e.g.,slow the progression of, arrest, and/or reverse) tissue calcification inthe subject, e.g., a subject with diabetes or chronic kidney disease.

Without wishing to be bound by the theory, oxidative stress induced bythe retention of uremic toxins depletes vascular endothelium offunctional Vitamin K₂ leading to suboptimal concentrations of certainVitamin K-dependent proteins, including carboxylated MGP and activatedProtein C. The subsequent reduced calcium binding capacity of these andother vitamin-dependent proteins contributes to abnormal tissuecalcification. High doses of substantially pure MK-7 and/or MKH2-7 canbe used to increase carboxylation of Vitamin K-dependent proteins,thereby preventing, slowing the progression of, arresting, and/orreversing tissue calcification, wherein the MK-7 and/or MKH2-7 areadministered in the form of a pharmaceutical composition. The MK-7and/or MKH2-7 can be administered in a single dosage unit (for example,a single capsule) or in multiple dosage units (for example, multiplecapsules) provided that the requisite amount of MK-7 and/or MKH2-7 isadministered per day. In certain embodiments, the pharmaceuticalcomposition comprises MK-7. In certain embodiments, the pharmaceuticalcomposition comprises MKH2-7. In certain embodiments, the pharmaceuticalcomposition comprises a combination of MK-7 and MKH2-7.

I. Menaquinone-7 (MK-7) and/or Menaquinol (MKH2-7)

The compositions of the invention comprise menaquinone-7 (MK-7), a formof Vitamin K₂. The IUPAC name for MK-7 is as2-[(2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyloctacosa-2,6,10,14,18,22,26-heptaenyl]-3-methylnaphthalene-1,4-dione,and the chemical structure of MK-7 is shown in Formula I.

Also contemplated for use in accordance with the methods herein ismenaquinol-7 (MKH2-7), which is a reduced form of MK-7. The IUPAC namefor MKH2-7 is2-[(2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyloctacosa-2,6,10,14,18,22,26-heptaenyl]-3-methylnaphthalene-1,4-diol,and the chemical structure of MKH2-7 is set forth in Formula II:

Without wishing to be bound by the theory, it is believed thatsupplemental MK-7 and/or MKH2-7 forms of Vitamin K provide increasedprotection against non-specific oxidation of Vitamin K intermediatescaused by uremia and/or dialysis.

The long aliphatic chain of MK-7 is not synthesized by humans but issynthesized in the colon by bacteria. Also, dietary sources of MK-7include bacterially fermented foods such as nattō (soy beans fermentedby Bacillus subtilis), cheeses, sauerkraut and buttermilk and pork, eel,plaice, and buckwheat bread. In addition, MK-7 is available as anutritional supplement (e.g., Vitamin K2 MK-7 from RejuvenationTherapeutics®; Bio-Tech™ Pharmacal MK-7 Vitamin K2, Fayetteville AR;NOW® Foods MK-7 Vitamin K-2).

The invention, however, employs substantially pure MK-7 and/or MKH2-7,administered in the form of a pharmaceutical composition.

MK-7 can also be produced synthetically, for example, as described inBaj et al. (2016) “Convergent Synthesis of Menaquinone-7 (MK-7),” ORG.PROCESS RES. DEV. 20:1026-1033, WO2010/034999 and WO2010/035000 (alsopublished as U.S. Patent Application No. 2011/0207967).

MKH2-7 can be synthesized from MK-7 by reducing MK-7 using conventionalreduction reactions known in the art, including, for example, byreduction with zinc and acetic acid as described by Marchand et al.(1991) “Mild and Highly Selective Ultrasound-promoted Zinc/Acetic AcidReduction of C═C Bonds in α,β-Unsaturated γ-Dicarbonyl Compounds,”SYNTHESIS 1991(3):198-200.

In this method, MK-7 (e.g., 1 g, 1.54 mmol) is dissolved in acetic acidglacial (15 mL) and powdered zinc (e.g., 0.8 g, 12.3 mmol) is added. Theresulting mixture is sonicated for 0.5 hour or the reaction can berefluxed until completion. The resulting mixture is filtered and theresidue is washed with dichloromethane. The combined filtrates areconcentrated in vacuo, to produce the pure reduction product. Theprocedure is performed under Argon. To stabilize MKH2-7 in the reducedform it may be helpful to admix MKH2-7 with one or more antioxidantssuch as vitamin C, a vitamin C ester (e.g., ascorbyl palmitate), and/orvitamin E, e.g., within a capsule or softgel. Alternatively, prodrugs ofMKH2-7 can be created, for example, where one or both of the hydroxylgroups are esterified with various groups (for example, acetate) toproduce a stable prodrug that is metabolized to produce MKH2-7 in thesubject.

II. Dosage Forms and Administration

In certain embodiments, compositions useful in accordance with theinvention comprise, consist essentially of, or consist of substantiallypure MK-7 and/or MKH2-7. Substantially pure refers to a composition ofactive ingredient comprising at least 95%, at least 96%, at least 97%,at least 98%, at least 99% or at least 99.5% by weight MK-7 and/orMKH2-7. In certain embodiments, MK-7 and/or MKH2-7 are the sole vitaminsadministered in a dosage form.

As used herein, the phrases “effective amount” and “therapeuticallyeffective amount” refer to the amount of a compound (e.g., MK-7 and/orMKH2-7) sufficient to effect one or more beneficial or desired results.An effective amount can be administered in one or more administrations,applications or dosages and is not intended to be limited to aparticular formulation or administration route.

In another embodiment, the invention provides a dosage form, forexample, an oral dosage form, comprising an effective amount of MK-7and/or MKH2-7, for example, from about 2 mg to about 1,000 mg, fromabout 2 mg to about 750 mg, from about 2 mg to about 500 mg, from about2 mg to about 250 mg, from about 2 mg to about 200 mg, from about 2 mgto about 150 mg, from about 2 mg to about 100 mg, from about 2 mg toabout 50 mg, from about 2 mg to about 25 mg, from about 5 mg to about1,000 mg, from about 5 mg to about 750 mg, from about 5 mg to about 500mg, from about 5 mg to about 250 mg, from about 5 mg to about 200 mg,from about 5 mg to about 150 mg, from about 5 mg to about 100 mg, fromabout 5 mg to about 50 mg, from about 5 mg to about 25 mg, from about 10mg to about 1,000 mg, from about 10 mg to about 750 mg, from about 10 mgto about 500 mg, from about 10 mg to about 250 mg, from about 10 mg toabout 200 mg, from about 10 mg to about 150 mg, from about 10 mg toabout 100 mg, from about 10 mg to about 50 mg, from about 10 mg to about25 mg, from about 15 mg to about 1,000 mg, from about 15 mg to about 750mg, from about 15 mg to about 500 mg, from about 15 mg to about 250 mg,from about 15 mg to about 200 mg, from about 15 mg to about 150 mg, fromabout 15 mg to about 100 mg, from about 15 mg to about 50 mg, from about15 mg to about 25 mg, from about 25 mg to about 1,000 mg, from about 25mg to about 750 mg, from about 25 mg to about 500 mg, from about 25 mgto about 250 mg, from about 25 mg to about 200 mg, from about 25 mg toabout 150 mg, from about 25 mg to about 100 mg, from about 25 mg toabout 50 mg, from about 50 mg to about 1,000 mg, from about 50 mg toabout 750 mg, from about 10 mg to about 500 mg, from about 50 mg toabout 250 mg, from about 50 mg to about 200 mg, from about 50 mg toabout 150 mg, from about 50 mg to about 100 mg, from about 75 mg toabout 1,000 mg, from about 75 mg to about 750 mg, from about 75 mg toabout 500 mg, from about 75 mg to about 250 mg, from about 75 mg toabout 200 mg, from about 75 mg to about 150 mg, from about 75 mg toabout 100 mg, from about 100 mg to about 1,000 mg, from about 100 mg toabout 750 mg, from about 100 mg to about 500 mg, from about 100 mg toabout 250 mg, from about 100 mg to about 200 mg, or from about 100 mg toabout 150 mg. In certain embodiments, 2 mg, 5 mg, 10 mg, 25 mg, 75 mg or100 mg of substantially pure MK-7 and/or MKH2-7 is administered to thesubject in a suitable dosage form, such as a tablet, caplet or capsule(e.g., a liquid or gel capsule).

In certain embodiments, the methods can include administering from about2 mg to about 100 mg of MK-7 and/or MKH2-7 to the subject per day. Inother embodiments, the method can include administering from about 2.5mg to about 100 mg, from about 3 mg to about 100 mg, from about 4 mg toabout 100 mg, or from about 5 mg to about 100 mg of MK-7 and/or MKH2-7to the subject per day. In certain embodiments, the method can includeadministering about 2 mg, 2.5 mg, about 3 mg, about 4 mg, about 5 mg,about or 7.5 mg of MK-7 and/or MKH2-7 to the subject per day. In otherembodiments, the method can include administering from about 10 mg toabout 100 mg of MK-7 and/or MKH2-7 to the subject per day, e.g.,administering 10, 25, 50, 75 or 100 mg of MK-7 and/or MKH2-7 to thesubject per day.

It is understood that the MK-7 and/or MKH2-7 can be administered in asingle dosage unit (e.g., 1 capsule) or in multiple (e.g., 2, 3, or 4,etc.) dosage units (e.g., multiple capsules). A composition for use inaccordance with the invention can be formulated as one or more dosageunits. Such dosage units may be administered once a day or a plurality(e.g., 1 to about 10, 1 to about 8, 1 to about 6, 1 to about 4 or 1 to2) of times per day, or as many times as needed to elicit a therapeuticresponse. The dosage units can be packaged in a kit, for example, a kitcontaining one or more blister packages of about 1 to about 20 dosageunits (e.g., capsules) per sheet or a bottle containing a plurality ofdosage units (e.g., capsules).

In certain embodiments, the pharmaceutical composition containing theMK-7 and/or MKH2-7 can be formulated for administration in solid orliquid form, including drenches (aqueous or non-aqueous solutions orsuspensions), tablets (e.g., those targeted for buccal, sublingual,and/or systemic absorption), boluses, powders, granules, pastes forapplication to the tongue, and/or topical creams. As used herein, theterm “pharmaceutical composition” refers to the combination of an activeagent with one or more pharmaceutically acceptable carriers (inert oractive) making the composition especially suitable for diagnostic ortherapeutic use in vivo or ex vivo. As used herein, the term“pharmaceutically acceptable carrier” refers to any of the standardpharmaceutical carriers and excipients, such as a phosphate bufferedsaline solution, water, emulsions (e.g., such as an oil/water orwater/oil emulsions), and various types of wetting agents. Thecompositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants, see Martin, Remington'sPharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa. (1975).

In certain embodiments, the MK-7 and/or MKH2-7 composition can beformulated in an oil, such as castor oil, sesame oil, medium chaintriglyceride (MCT) oil, olive oil, soybean oil, or coffee bean oil.Excipients suitable for use with the MK-7 and/or MKH2-7 compositioninclude antioxidants, bioavailability enhancers, solubility enhancers orsolubilizers, stabilizers, etc.

In certain embodiments, the MK-7 and/or MKH2-7 composition includes oneor more antioxidants such as one or more antioxidants selected fromDL-alpha tocopherol (vitamin E), ascorbic acid (vitamin C) or a vitaminC ester, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA),lecithin, citric acid, sesame oil, olive oil, soybean oil, and coffeebean oil.

In certain embodiments, the MK-7 and/or MKH2-7 composition includes anemulsifier selected from Tween® 20 (polyethylene glycol sorbitanmonolaurate), Tween® 80 (polyethylene glycol sorbitan monooleate),Kolliphor® EL (polyoxyl castor oil, also called Cremophor EL), Capmul®MCM (mono-diglyceride of medium chain fatty acids such as caprylic andcapric), lecithin, Lauroglycol™ 90 (propylene glycol monolaurate (typeII)), deoxycholic acid, Phosal® 50 PG (lecithin in propylene glycol,sunflower mono-diglycerides, ascorbyl palmitate), beeswax, and waxflakes.

In certain embodiments, the MK-7 and/or MKH2-7 composition includes abioavailability enhancer selected from Maisine® CC (glycerylmonolinoleate), Gelucire® 44/14 (lauroyl polyoxyl-32 glycerides),Capmul® PG-8 NF (propylene glycol monoester of caprylic acid), Plurol®Oleique (polyglyceryl-6 dioleate), Lauroglycol™ 90 (propylene glycolmonolaurate (type II), Labrasol® (caprylocaproyl polyoxyl-8 glycerides),Captisol® (SBE-beta-cyclodextrin), Labrafil® M2125CS (linoleoylPolyoxyl-6 glycerides), and Phosal® 50 PG (lecithin in propylene glycol,sunflower mono-diglycerides, ascorbyl palmitate).

In certain embodiments, the MK-7 and/or MKH2-7 composition includes asolubility enhancer or solubilizer selected from oleic acid, Kolliphor®EL (polyoxyl castor oil, also called Cremophor EL), Vitamin E TPGS(D-α-tocopherol polyethylene glycol-1000 succinate), Maisine® CC(glyceryl monolinoleate), Gelucire® 44/14 (lauroyl polyoxyl-32glycerides), Miglyol® 812N (esters of saturated coconut and palm kerneloil-derived caprylic fatty acids and glycerin), Plurol® Oleique(Polyglyceryl-6 Dioleate), Lauroglycol™ 90 (propylene glycol monolaurate(type II), Labrasol® (Caprylocaproyl polyoxyl-8 glycerides), Kolliphor®EL (polyoxyl castor oil), Captisol® (SBE-beta-cyclodextrin), Encapsin™HPB (hydroxypropyl-beta-cyclodextrin), Peceol™ (glycerol/glycerylmonooleate (type 40)), sodium deoxycholate, deoxycholic acid, Labrafil®M2125CS (linoleoyl Polyoxyl-6 glycerides), and medium-chain mono- anddiglycerides.

In certain embodiments, the MK-7 and/or MKH2-7 composition includes asolvent selected from the group consisting of isopropyl myristate,Capmul® PG-8 NF (propylene glycol monoester of caprylic acid),Lauroglycol™ 90 (propylene glycol monolaurate (type II)), Polyethyleneglycol (PEG), and propylene glycol.

In certain embodiments, the MK-7 and/or MKH2-7 composition includes astabilizer selected from the group consisting of oleic acid and DL-alphatocopherol.

In certain embodiments, the MK-7 and/or MKH2-7 composition includes asurfactant selected from the group consisting of sodium oleate, Span 20(sorbitan laurate), Span® 80 (sorbitan oleate), Vitamin E TPGS(D-α-tocopherol polyethylene glycol-1000 succinate), Lauroglycol™ 90(propylene glycol monolaurate (type II)), Labrasol® (Caprylocaproylpolyoxyl-8 glycerides), polyethylene glycol (PEG), and Captisol®(SBE-beta-cyclodextrin).

Other suitable excipients include one or more fatty acids selected fromlauric, valeric, caproic, capric, caprylic, myristic, palmitic,palmitoleic, stearic, and arachidic acid and their esters.

In certain embodiments, compositions useful in the practice of theinvention are orally deliverable. The terms “orally deliverable” or“oral administration” herein include any form of delivery of thecompositions described herein to a subject, wherein the composition isplaced in the mouth of the subject, whether or not the composition isswallowed. Thus “oral administration” includes buccal and sublingual aswell as esophageal administration.

In certain embodiments, the MK-7 and/or MKH2-7 is administered to thesubject for at least 2 weeks, e.g., 2, 3, 4, 5 weeks, or more. Incertain embodiments, the MK-7 and/or MKH2-7 is administered for at least6 weeks. In certain embodiments, the MK-7 and/or MKH2-7 is administeredto a subject for at least 2 months, at least 3 months, at least 6months, at least 1 year, at least 2 years, at least 3 years, orindefinitely. In certain embodiments, the MK-7 and/or MKH2-7 isadministered at least as long as the subject is receiving ananticoagulant therapy that is not warfarin or a drug having a similarmechanism of action as warfarin (e.g., acenocoumarol and phenprocoumon)and/or statin therapy. If the subject is undergoing hemodialysis, theMK-7 and/or MKH2-7 can be administered to the subject for a period thatincludes the duration of hemodialysis.

In certain embodiments, the MK-7 and/or MKH2-7 composition isadministered together with a statin such as simvastatin, lovastatin,atorvastatin, pravastatin, pitavastatin, rosuvastatin, and fluvastatin.The MK-7 and/or MKH2-7 composition can be formulated into a singledosage form, such as a dosage form discussed herein, with the statin. Inother embodiments, the MK-7 and/or MKH2-7 composition is formulated intoa separate dosage form from the statin. Formulation of statins, such assimvastatin, lovastatin, atorvastatin, pravastatin, pitavastatin,rosuvastatin, and fluvastatin, is well known in the art.

As used herein, the terms “subject” and “patient” are usedinterchangeably and refer to an organism to be treated by the methodsand compositions of the present invention. Such organisms are preferablymammals (e.g., human, mouse, rat, guinea pig, dog, cat, horse, cow, pig,or non-human primate, such as a monkey, chimpanzee, baboon, and rhesus),and more preferably humans.

III. Methods

The disclosure relates, in part, to a method of preventing or treating,for example, by slowing the progression of, arresting, and/or reversingtissue calcification in a subject in need thereof. In certainembodiments, the method relates to preventing tissue calcification. Incertain embodiments, the method relates to slowing the progression oftissue calcification. In certain embodiments, the method relates toarresting tissue calcification. In certain embodiments, the methodrelates to reversing tissue calcification.

In certain embodiments, the disclosure relates to a method of improvingaortic compliance in a subject in need thereof, the method comprisingadministering to the subject an effective amount of substantially puremenaquinone-7 (MK-7) and/or menaquinol-7 (MKH2-7) per day. In certainembodiments, the disclosure relates to a method of peventing, slowingthe progression of, arresting and/or reversing peripheral vasculopathyin a subject in need thereof, wherein the subject has ESRD or CKD, themethod comprising administering to the subject an effective amount ofsubstantially pure menaquinone-7 (MK-7) and/or menaquinol-7 (MKH2-7) perday.

As used herein, the term “treating” includes any effect, for example,lessening, reducing, slowing the progression of, arresting, modulating,ameliorating or eliminating, that results in the improvement of thecondition, disease, disorder, and the like, or ameliorating a symptomthereof. Treating can be curing, improving, or at least partiallyameliorating the disorder. In certain embodiments, treating is curingthe disease. The term “disorder” refers to and is used interchangeablywith, the terms disease, condition, or illness, unless otherwiseindicated.

The method can include administering to the subject a menaquinone-7(MK-7) and/or menaquinol-7 (MKH2-7) composition as described herein(e.g., at least 2 mg of substantially pure MK-7 and/or MKH2-7 per day,e.g., 10, 15, 25, 50, 75 mg or more per day), thereby to prevent ortreat (e.g., slow the progression of, arrest, and/or reverse) tissuecalcification. Tissue calcification refers to the accumulation ofcalcium salts in a body tissue. In certain embodiments, the tissuecalcification is a soft tissue calcification. The term “soft tissue”includes any tissue that is not a bone or a tooth. In certainembodiments, the tissue calcification is a vascular or dermal (skin)calcification.

1. Diabetes, CKD, ESRD, Hemodialysis

In certain embodiments, the subject to be treated with the methodsand/or compositions described herein has diabetes, e.g., type I or IIdiabetes, or has been diagnosed with pre-diabetes. Diabetes often leadsto chronic kidney disease (CKD) due to uncontrolled blood sugar andblood pressure damaging capillaries in the kidney. CKD often leads tothe development of uremia (urea in the blood), which can inhibit cyclicregeneration of Vitamin K, leading to tissue calcification. Accordingly,in certain embodiments, the subject has CKD.

Patients with chronic kidney disease are classified into stages basedupon their glomerular filtration rate (GFR). The GFR for stage 1patients is ≥90 mL/minute/1.73 m², stage 2 is 60-89 mL/minute/1.73 m²,stage 3 is 30-59 mL/minute/1.73 m², stage 4 is 15-29 mL/minute/1.73 m²,and stage 5 is <15 mL/minute/1.73 m². In certain embodiments, thesubject has stage 3, stage 4, or stage 5 CKD. Patients with stage 4 orstage 5 CKD are considered to have end stage renal disease (ESRD). Othertypes of kidney disease, such as polycystic kidney disease, also canlead to ESRD. In certain embodiments, the subject has ESRD.

Patients with ESRD require hemodialysis. However, hemodialysis oftenleads to the oxidation of numerous tissue proteins, and both CKD andESRD patients often exhibit a higher percentage (15-fold) of carbonylproteins compared to normal controls. The percentage of carbonylproteins may be even higher among patients receiving hemodialysis,suggesting that hemodialysis contributes to oxidative burden.

As discussed supra, the oxidative load generated by hemodialysis isbelieved to lead to the oxidation of KH2 back to MK-7. It iscontemplated that the oxidation of KH2 by hemodialysis blocks itsability to function as a co-factor for GGCX leading to reduced gammacarboxylation of Vitamin K-dependent proteins. Thus, the oxidativeeffects of hemodialysis may contribute to the high rates of soft tissue(e.g., vascular) calcification observed within the CKD and ESRDpopulations. Accordingly, in certain embodiments herein, the subject isreceiving hemodialysis.

2. Anticoagulant Therapy

Vitamin K is necessary for the production of clotting factors. However,certain anticoagulants, for example, certain oral anticoagulants, act asVitamin K antagonists to prevent blood clotting. Given that tissuecalcification can occur when Vitamin K activity is dysregulated, themethods herein relate to preventing or treating (e.g., slowing theprogression of, arresting, and/or reversing) tissue calcification in asubject receiving anticoagulant therapy, and in need thereof, comprisingadministering to the subject a menaquinone-7 (MK-7) and/or menaquinol-7(MKH2-7) containing composition as described herein, thereby to preventor treat (e.g., slow the progression of, arrest, and/or reverse) tissuecalcification.

Exemplary anti-coagulant therapies include inhibitors of Factor Xaactivity or Factor IIa activity. Inhibitors of Factor Xa activityinclude the oral therapies apixaban (e.g., Eliquis®, Bristol-MyersSquibb), rivaroxaban (e.g., Xarelto®, Janssen), betrixaban (e.g.,Bevyxxa®, Portola Pharmaceuticals), and edoxaban (e.g., Savaysa®,Daiichi Sankyo), and the subcutaneous therapy fondaparinux (e.g.,Arixtra®, GlaxoSmithKline). Inhibitors of Factor IIa (thrombin) activityinclude the oral therapies dabigratran (e.g., Pradaxa®, BoehringerIngelheim) and the intravenous therapy argatroban (e.g., Pfizer).

In certain embodiments, the subject is simultaneously receiving anon-warfarin-based anticoagulant therapy. Non-warfarin-basedanticoagulant therapies can be based on an anticoagulant that does notprimarily act to block the conversion of Vitamin K to Vitamin Khydroquinone and/or the conversion of Vitamin K 2,3-epoxide back toVitamin K. Exemplary non-warfarin-based anticoagulants may act as ainhibitor of Factor Xa activity or an inhibitor of Factor IIa activity.Exemplary non-warfarin-based anticoagulants include apixaban,rivaroxaban, betrixaban, edoxaban, dabigratran, fondaparinux, andargatroban. In certain embodiments, the subject has previously beenexposed to warfarin-based anti-coagulation therapy.

In certain embodiments, the methods described herein relate topreventing or treating (e.g., slowing the progression of, arresting,and/or reversing) tissue calcification in a subject in need thereof,wherein the subject has CKD or ESRD and is undergoing oral,non-warfarin-based anticoagulant therapy. The method can compriseadministering to the subject at least 2 mg of substantially puremenaquinone-7 (MK-7) and/or menaquinol-7 (MKH2-7) per day, thereby toprevent or treat (e.g., slow the progression of, arrest, or reverse)tissue calcification. The subject may also have diabetes, pre-diabetes,and/or may be undergoing hemodialysis.

3. Statin Therapy

It has been observed that coronary artery calcification can be increasedupon statin use (Saremi et al. (2012) “Progression of VascularCalcification Is Increased With Statin Use in the Veterans AffairsDiabetes Trial (VADT),” DIABETES CARE 35:2390-2392). Lipophilic statinshave been shown to inhibit the enzymatic activity of UbiAprenyltransfease domain-containing protein (UBIAD1), an enzyme thatplays a role in Vitamin K synthesis (Nakagawa et al. (2010)“Identification of UBIAD1 as a novel human menaquinone-4 biosyntheticenzyme,” NATURE 468(7320):117-21). Further, in vitro experiments appearto demonstrate that Vitamin K synthesis is impaired in the presence ofstatins (Chen et al. (2017) “Does statins promote vascular calcificationin chronic kidney disease?” EUR. J. CLIN. INVEST. 47(2): 137-148).

Without wishing to be bound by the theory, it is contemplated that HMGcoA reductase inhibitors directly inhibit MK-4 production in coronaryvascular smooth muscle, which may explain why statins have not beenshown to reduce cardiovascular mortality in CKD or ESRD patients. Onepossible explanation for this observation is that CKD and ESRD patientsare functionally Vitamin K deficient, and that blocking endogenousvascular smooth muscle cell vitamin K (MK-4) production only serves toworsen the calcification of the media of the vessel, thus mitigating anypotential benefits of lipid reduction. Moreover, the loss of vascularcompliance accompanying the increase in calcification may contribute tooverall cardiovascular mortality.

Accordingly, provided herein is a method for preventing or treating(e.g., slowing the progression of, arresting, and/or reversing) tissuecalcification in a subject in need thereof, wherein the subject isreceiving a statin. The method can include administering to the subjecta menaquinone-7 (MK-7) and/or menaquinol-7 (MKH2-7) composition asdescribed herein. In certain embodiments, the subject is receiving astatin, such as simvastatin (e.g., Zocor®, Merck & Co., Inc.),lovastatin (e.g., Mevacor®, Merck & Co., Inc.), atorvastatin (e.g.,Lipitor®, Pfizer), pravastatin (e.g., Pravachol®, Bristol-Myers SquibbCo.), pitavastatin (e.g., Livalo®, Kowa Pharmaceuticals America),rosuvastatin (e.g., Crestor®, AstraZeneca), and fluvastatin (e.g.,Lescol®, Novartis Pharmaceuticals).

Further provided herein is a method for preventing or treating (e.g.,slowing the progression of, arresting, and/or reversing) tissuecalcification in a subject in need thereof, wherein the method includesadministering to the subject menaquinone-7 (MK-7) and/or menaquinol-7(MKH2-7) composition as described herein and a statin, such assimvastatin, lovastatin, atorvastatin, pravastatin, pitavastatin,rosuvastatin, and fluvastatin. The menaquinone-7 (MK-7) and/ormenaquinol-7 (MKH2-7) and statin can be administered as separate dosageforms, or in the same dosage form.

4. Improving Aortic Compliance and Stopping and/or Reversing PeripheralVasculopathy

In certain embodiments, the disclosure relates to a method of arrestingand/or reversing peripheral vasculopathy in a subject in need thereof,wherein the subject has ESRD or CKD, the method comprising administeringto the subject an effective amount of substantially pure menaquinone-7(MK-7) and/or menaquinol-7 (MKH2-7). In certain embodiments, thedisclosure relates to a method of improving aortic compliance in asubject in need thereof, the method comprising administering to thesubject an effective amount of substantially pure menaquinone-7 (MK-7)and/or menaquinol-7 (MKH2-7).

The term “vasculopathy” refers to any disease or disorder affectingblood vessels. It can include any inflammatory, metabolic, coagulative,embolic or degenerative disease, disorder, or condition. In certainembodiments, the vasculopathy is vascular calcification, e.g., medial orintimal vascular calcification.

One of the clinical consequences of vascular calcification is arterialstiffness, which leads to a decrease in vascular compliance (e.g.,vascular elasticity), e.g., in aortic compliance. Thus, in accordancewith the methods disclosed herein, administration of an effective amountof substantially pure menaquinone-7 (MK-7) and/or menaquinol-7 (MKH2-7)may improve aortic compliance. Reduction in aortic compliance can beassessed using aortic plethysmography (i.e., the capacity of a bloodvessel to dilate), see, e.g., Inuma et al. (2012) HONG KONG JOURNAL OFNEPHROLOGY 14(2):48-53. Reduction in aortic compliance also can beassessed by measuring pulse wave velocity (PWV). PWV is the velocity atwhich the arterial pulse moves through the circulatory system and isused as a measure of arterial stiffness. Higher PWV corresponds tohigher arterial stiffness, and therefore lower vascular compliance.Methods for measuring PWV are known in the art and described, forexample, by Pereira et al. (2015) “Novel Methods for Pulse Wave VelocityMeasurement,” J. MED. BIOL. ENG. 35:555-565. Briefly, PWV can beassessed by measuring regional PWV, such as carotid-femoral PWV, orlocal PWV. Methods for measuring regional PWV are known in the art andinclude, for example, the use of a pulse transducer probe, TY-360pressure transducer, doppler unit synchronism with electrocardiogram(ECG), pulse transducer probes, photoplethysmography, Complior®(piezoelectric pressure transducers), SphygmoCor®, Arteriograph®, andPulsePen® (tonometer and integrated electrocardiogram unit). Methods formeasuring local PWV are known in the art and include, for example,magnetic resonance imaging, ultrasound, and angiography.

In certain embodiments, administration of the MK-7 and/or MKH2-7increases aortic compliance of a blood vessel by at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100%. In certain embodiments, administration of the MK-7and/or MKH2-7 increases aortic compliance of a blood vessel by about10%-500%, by about 50%-200%, by about 75%-125%. In certain embodiments,administration of the MK-7 and/or MKH2-7 increases aortic compliance ofa blood vessel by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to avessel prior to administration of the MK-7 and/or MKH2-7. In certainembodiments, administration of the MK-7 and/or MKH2-7 increases aorticcompliance of a blood vessel by about 10%-500%, by about 50%-200%, byabout 75%-125% relative to a vessel prior to administration of the MK-7and/or MKH2-7.

Coronary arterial calcium scores (CAC) can be used to estimate theextent of calcification of thoracic arteries. A high CAC score isindicative of calcification, and treatment has the aim of arresting thelong term increase in CAC score, or reversing it, or slowing the rate ofincrease.

5. Chronic Obstructive Pulmonary Disease

Chronic Obstructive Pulmonary Disease is a term used to describeprogressive lung disease that makes breathing difficult. The two primaryforms of COPD are emphysema and chronic bronchitis. Symptoms of COPDinclude difficulty breathing, cough, mucus production, and wheezing. Inaddition, elastinolysis (proteolysis of elastin) is a key feature ofCOPD. It contributes to the loss of arterial flexibility and promotescalcification of the intimal media of blood vessels. It also has beenshown to be a strong predictor of mortality in COPD patients (Rabinovichet al., (2016) “Circulating desmosine levels do not predict emphysemaprogression but are associated with cardiovascular risk and mortality inCOPD,” ERJ Express doi: 10.1183/13993003.01824-2015). MGP has beendemonstrated to inhibit the production of matrix metalloproteases thatpromote elastinolysis. Therefore, it has been hypothesized that VitaminD is a critical determinant of the rate of elastin degradation, and thatlow Vitamin D levels lead to low MGP activity that is inadequate toprotect from elastinolysis (Piscaer et al., (2017) “Vitamin Ddeficiency: the linking pin between COPD and cardiovascular diseases?”RESP. RES. 18:189). Without wishing to be bound by the theory, enhancedproduction of activated (carboxylated) MGP by administration of MK-7and/or MKH2-7 can act to suppress the deleterious effects ofelastinolysis in a subject having COPD thereby to prevent, or slow theprogression of, arrest, or reverse the one or more symptoms of COPD.

Reduction of symptoms of COPD, including difficulty breathing, cough,mucus production, and wheezing, can be measured by any means known inthe art. A slowing of the rate of increase of one or more COPD symptomsover time is indicative of a slowing of the progression of the one ormore symptoms of COPD, or a reduction in one or more symptoms, such asby 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100% is indicative of a reversal of the oneor more symptoms of COPD.

Elastinolysis can be measured by any method known in the art, including,for example, by measuring blood isodesmosine levels in a subject. Aslowing of the rate of increase in isodesmosine levels over time isindicative of a slowing of the progression of the one or more symptomsof COPD, or a reduction in isodesmosine levels, such as by 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100% is indicative of a reversal of the one or moresymptoms of COPD.

IV. Biomarkers Useful for Determining the Prevention, Slowing theProgression of, Arresting or Reversing Tissue Calcification in a Subject

In certain embodiments, biomarkers can be assessed to determine whetherthe methods and/or compositions described herein are effective atpreventing or treating (e.g., slowing the progression of, arresting,and/or reversing) tissue calcification in a subject. These include, forexample, measuring a subject's T50 times and measuring a ratio ofcarboxylated Vitamin K-dependent proteins to non-carboxylated proteins,as described in more detail below.

1. Primary Calciprotein Particles (CPPs) and Serum T50

Under normal physiologic conditions, pathological calcification isprevented in part by primary calciprotein particles (CPPs) (Price, etal., supra). CPPs comprise circulating calcium phosphate crystalscomplexed with two calcification inhibiting proteins, Fetuin A andMatrix Gla Protein. (Id.) Matrix Gla Protein is a Vitamin K-dependentprotein and formation of CPPs is dependent upon the gamma carboxylationof Matrix Gla Protein. (Id.) Pre-clinical studies suggest that thecalciprotein system functions as an alternative means for preventingpathologic calcification when humoral lines of defense such aspyrophosphate, magnesium and albumin are overwhelmed. The binding ofcalcium-phosphate crystals by primary CPPs occurs in a coordinated andtime-dependent process.

The time necessary for 50% (T50) conversion of primary CPPs to secondaryCPPs is an accurate and highly sensitive means for determining thecapacity of plasma to sequester excess calcium phosphate crystals. Theconversion from primary to secondary forms involves the formation of amore elongated crystal. The elongation leads to a more turbid solutionwhich can be detected by nephelometry. Under conditions of heavy calciumphosphate burden, the time to reach 50% conversion is shortened due toreduced reserves for calcium phosphate absorption. Thus, short T50 timessuggest a reduced capacity to absorb calcium phosphate crystals whereasprolonged T50 times are consistent with high capacities. Short T50 timesare often associated with warfarin treatment, or hemodialysis or uremicoxidative load, and generally are associated with increased risk ofmyocardial infarctions, heart failure and all-cause mortality.

Without wishing to be bound by theory, it is believed that theadministration of MK-7 and/or MKH2-7 increases T50 times and improves asubject's capacity to prevent pathologic calcification. Because subjectswith CKD and ESRD exhibit reduced levels of carboxylated Matrix GlaProtein, which is Vitamin K-dependent and essential for the formation ofprimary CPP, administration of MK-7 and/or MKH2-7 in accordance with themethods of the present disclosure can reduce the risk for pathologiccalcification and prevent the development of soft tissue (e.g.,vascular) calcification.

Accordingly, in certain embodiments of the methods disclosed herein,administration of the MK-7 and/or MKH2-7 to the subject increases thesubject's serum T50 value (e.g., by at least 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%) relative to the subject's serum T50 value prior to administrationof the MK-7 and/or MKH2-7. Methods for measuring a subject's serum T50value are known in the art. (See, e.g., Pasch et al. (2012)“Nanoparticle-based test measures overall propensity for calcificationin serum.” J. AM. SOC. NEPHROL. 23(10): 1744-52; Dahle et al., (2016)“Serum Calcification Propensity Is a Strong and Independent Determinantof Cardiac and All-Cause Mortality in Kidney Transplant Recipients.” AM.J. TRANSPLANT 16(1): 204-12; and Smith et al. (2014) “SerumCalcification Propensity Predicts All-Cause Mortality in PredialysisCKD,” J. AM. SOC. NEPHROL. 25(2):339-348.)

In certain embodiments, the disclosure relates to a method of increasinga serum T50 value in a subject having a disorder as described herein,the method comprising administering at least 2 mg/day (e.g., at least 2mg of substantially pure MK-7 and/or MKH2-7 per day, e.g., 10, 15, 25,50 or 75 mg per day) MK-7 and/or MKH2-7 to the subject, whereinadministration of the MK-7 and/or MKH2-7 to the subject increases thesubject's serum T50 value (e.g., by at least 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%) relative to the subject's serum T50 value prior to administrationof the MK-7 and/or MKH2-7.

2. Carboxylation of Vitamin K-Dependent Proteins

Despite dietary deficiencies, Vitamin K levels among ESRD patients maynot be reduced. For example, a study of 172 stable hemodialysis patientsfound that only 6% of patients exhibited a clinically significantdeficiency in Vitamin K. (Holden et al. (2010) “Vitamins K and D Statusin Stages 3-5 Chronic Kidney Disease,” CLIN. J. AM. SOC. NEPHROL.5:590-597.) However, when patients were examined for the level ofcarboxylated osteocalcin, a Vitamin K-dependent protein, approximately60% of patients had reduced levels. (Id.) Similar results were shown forother Vitamin K-dependent proteins, such as PIVKA-II. (Id., and Pilkeyet al. (2007) “Subclinical Vitamin K Deficiency in HemodialysisPatients,” AM. J. KIDNEY. DIS. 49:432-439.) These results are consistentwith the hypothesis that in uremic patients, total Vitamin K levels canbe normal while generation of reduced forms are blocked by the oxidativeproperties of uremia. (Id.)

The observation that oxidant conditions can disrupt the Vitamin K cyclesuggests that the oxidant load generated during hemodialysis furthercontributes to the high rates of soft tissue (e.g., vascular)calcification observed within the ESRD population. Delivery ofhemodialysis is known to lead to the oxidation of numerous tissueproteins, and both CKD and ESRD patients exhibit a higher percentage(15-fold) of carbonyl proteins compared to normal controls.

Without wishing to be bound by the theory, it is believed that theoxidative load generated by uremia and/or hemodialysis leads tooxidation of the functional Vitamin K hydroquinone (KH2) to thenon-functional native vitamin. The oxidation of KH2 blocks its abilityto function as a co-factor for GGCX which leads to reduced gammacarboxylation of Vitamin K-dependent proteins. Thus, administration ofMK-7 and/or MKH2-7 to subjects with diabetes, CKD, and/or ESRD and/orsubjects undergoing hemodialysis, can increase the ratio of one or morecarboxylated Vitamin K-dependent proteins to non-carboxylated VitaminK-dependent proteins in these subjects and/or decrease the amount of oneor more non-carboxylated Vitamin K-dependent proteins in these subjects.

Accordingly, in certain embodiments, administration of MK-7 and/orMKH2-7 increases a ratio of a carboxylated to a non-carboxylated VitaminK-dependent protein in the subject's plasma (e.g., by at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100%) relative to the ratio prior to administration ofthe MK-7 and/or MKH2-7. In certain embodiments, administration of MK-7and/or MKH2-7 increases a ratio of a carboxylated to a non-carboxylatedVitamin K-dependent protein in the subject's plasma by between about 10%and 500% or higher, by about 30% and about 500%, between about 30% andabout 200%, between about 30% and about 100%. In certain embodiments,administration of MK-7 and/or MKH2-7 increases a ratio of a carboxylatedto a non-carboxylated Vitamin K-dependent protein in the subject'splasma by between about 50% and about 500%, between about 50% and about200%, between about 50% and about 100%. In certain embodiments,administration of MK-7 and/or MKH2-7 increases a ratio of a carboxylatedto a non-carboxylated Vitamin K-dependent protein in the subject'splasma by between about 70% and about 500%, between about 70% and about200%, between about 70% and about 100%.

In certain embodiments, administration of MK-7 and/or MKH2-7 reduces anamount of non-carboxylated Vitamin K-dependent protein in the subject'splasma (e.g., by at least 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) relative to theamount prior to administration of the MK-7 and/or MKH2-7. In certainembodiments, administration of MK-7 and/or MKH2-7 reduces an amount of anon-carboxylated Vitamin K-dependent protein in the subject's plasma bybetween about 5% and about 100%, between about 5% and about 80%, betweenabout 5% and about 30%, between about 20% and about 100%, or betweenabout 20% and about 100%.

In certain embodiments, the disclosure relates to a method forincreasing a ratio of a carboxylated to a non-carboxylated VitaminK-dependent protein in the plasma of a subject having a disorder asdescribed herein, the method comprising administering at least 2 mg/day(e.g., at least 2 mg of substantially pure MK-7 and/or MKH2-7 per day,e.g., 10, 15, 25, 50 or 75 mg per day) MK-7 and/or MKH2-7 to thesubject, wherein administration of the MK-7 and/or MKH2-7 to the subjectincreases a ratio of a carboxylated to a non-carboxylated VitaminK-dependent protein in the subject's plasma (e.g., by at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100%) relative to the ratio prior to administration ofthe MK-7 and/or MKH2-7.

In certain embodiments, the disclosure relates to a method fordecreasing an amount of a non-carboxylated Vitamin K-dependent proteinin the plasma of a subject having a disorder as described herein, themethod comprising administering at least 2 mg/day (e.g., at least 2 mgof substantially pure MK-7 and/or MKH2-7 per day, e.g., 10, 15, 25, 50or 75 mg per day) MK-7 and/or MKH2-7 to the subject, whereinadministration of the MK-7 and/or MKH2-7 to the subject decreases anamount of a non-carboxylated Vitamin K-dependent protein in thesubject's plasma (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%)relative to the amount prior to administration of the MK-7 and/orMKH2-7.

In certain embodiments, the Vitamin K-dependent protein is selected fromMatrix Gla Protein (MGP), Growth Arrest Specific Gene 6 (Gas-6) protein,PIVKA-II protein, osteocalcin, activated Protein C, or activated ProteinS. Carboxylated forms of MGP, Gas-6, PIVKA-II, osteocalcin, activatedProtein C and activated Protein S can be measured using sandwich assays,as is known in the art. (See, e.g., Nigwekar et al. (2017) “VitaminK-Dependent Carboxylation of Matrix Gla Protein Influences the Risk ofCalciphylaxis,” J. AM. SOC. NEPHROL. 28:1717-1722 (detecting MGP);Gla-Type Osteocalcin (GLa-OC) ELISA Kit (Takara, Mountain View,Calif.)); STA-Staclot Protein C (Stago, Asnières sur Seine, France; andREAADS Protein S Antigen, cat. no. K036-001, Diapharma Group, WestChester, Ohio). An increase in the ratio of carboxylated tonon-carboxylated Vitamin K-dependent proteins in a subject followingadministration with MK-7 is indicative of successful rescue of the GGCXcarboxylation system. Accordingly, effective MK-7 and/or MKH2-7administration can lead to an increase in the ratio of carboxylated tonon-carboxylated proteins in the subject.

PIVKA-II is an abnormal form of prothrombin which has not beencarboxylated, also known as des-gamma carboxyprothrombin (DCP). Thus,elevated levels of PIVKA-II correspond to poorer vitamin K status. As abiomarker of vitamin K status, PIVKA-II has the advantage of beingindependent of kidney function and lipid profile (Elliot et al. (2014)“Assessment of potential biomarkers of subclinical vitamin K deficiencyin patients with end-stage kidney disease,” CAN. J. KIDNEY HEALTH DIS.1:13.) According to the methods herein, measuring PIVKA-II in a subjectis indicative of successful rescue of the GGCX carboxylation system.PIVKA-II plasma levels can be measured by any method known in the art,e.g., using ELISA (Diagnostica Stage, Parsippany, N.J.). (See also,e.g., Dituri et al. (2012) “PIVKA-II plasma levels as markers ofsubclinical vitamin K deficiency in term infants,” J. MATERNAL-FETAL &NEONATAL MEDICINE 25(9): 1660-1663.)

MGP is believed to be a potent calcification inhibitor of the arterialwall and other soft tissues in non-human animals, where its activitydepends on vitamin K-dependent γ-glutamate carboxylation (Schurgers etal. (2007a) “Post-translational modifications regulate matrix Glaprotein function: importance for inhibition of vascular smooth musclecell calcification,” J. THROMB. HAEMOST. 5:2503-2511; Westenfeld et al.(2012) “Effect of vitamin K2 supplementation on functional vitamin Kdeficiency in hemodialysis patients: a randomized trial,” AM. J. KIDNEYDIS. 59(2):186-195; Schurgers et al. (2013) “Vitamin K-dependentcarboxylation of matrix Gla-protein: a crucial switch to control ectopicmineralization,” TRENDS MOL. MED. 2013;19(4):217-26; Marles et al.(2017) “US Pharmacopeial Convention safety evaluation of menaquinone-7,a form of vitamin K,” NUTR. REV. 75(7):553-578). Uncarboxylated MGP,formed as a result of vitamin K deficiency, is associated withcardiovascular disease. Recent studies suggest poor vitamin K status inhemodialysis patients (Westenfeld et al. (2012), supra). The circulatinginactive form of MGP (dp-ucMGP) was shown to increase progressively inpatients with CKD (Schurgers et al. (2010) “The Circulating InactiveForm of Matrix Gla Protein Is a Surrogate Marker for VascularCalcification in Chronic Kidney Disease: A Preliminary Report,” CLIN J.AM. SOC. NEPHROL. 5(4):568-75) and to be predictive of vitamin K statusand correlated with vascular calcification in patients on hemodialysis(Delanaye et al. (2014) “Dephosphorylated-uncarboxylated Matrix Glaprotein concentration is predictive of vitamin K status and iscorrelated with vascular calcification in a cohort of hemodialysispatients,” BMC NEPHROL. 15(1):145). Plasma dp-ucMGP has therefore beenproposed as a surrogate marker for vascular calcification in CKD(Schurgers et al. (2010), supra). In addition, lower levels ofcirculating desphospho-carboxylated MGP (dp-cMGP) was proposed as apredictor of mortality in hemodialysis patients (Schlieper et al. (2011)“Circulating nonphosphorylated carboxylated matrix gla protein predictssurvival in ESRD,” J. AM. SOC. NEPHROL. 22(2):387-95).

Osteocalcin is a non-collagenous bone matrix protein synthesized bymature osteoblasts, and involved in bone formation and re-gelation ofbone mineralization (Elliott et al. 2014, supra; Marles et al. 2017,supra). The proportion of osteocalcin that is uncarboxylated is asensitive marker of vitamin K status in bone, and subclinical vitamin Kdeficiency is defined by an increase in the proportion of uncarboxylatedosteocalcin above 20% (Elliott et al. 2014, supra). Supplementation withMK-7 at doses of 100-200 μg/d for 4 to 12 weeks was shown to increasethe ratio of carboxylated to undercarboxylated osteocalcin significantlyand in a dose-dependent manner (Inaba et al. (2015), “Low-Dose DailyIntake of Vitamin K(2) (Menaquinone-7) Improves Osteocalcinγ-Carboxylation: A Double-Blind, Randomized Controlled Trials,” J. NUTR.SCI. VITAMINOL 61(6):471-80).

Vitamin K-dependent γ-carboxyglutamate proteins are synthesized in theliver, and help maintain normal blood coagulation through a balance ofboth procoagulant factors (II, VII, IX, and X) and anticoagulantproteins (C and S) (Schurgers et al. (2007c) “Vitamin K-containingdietary supplements: comparison of synthetic vitamin K1 andnatto-derived menaquinone-7,” BLOOD 109(8):3279-83; Marles et al. 2017,supra). Protein C regulates the coagulation process by neutralizing theprocoagulant activities of factors V and VIII in the presence of thecofactor Protein S (Marlar et al. (2017) “Assessment of HereditaryThrombophilia: Performance of Protein C (PC) Testing,” METHODS MOL.BIOL. 1646:145-151). Hypercoagulable states, such as protein C and/orprotein S deficiencies, have been reported in patients withcalciphylaxis, and proposed as factors increasing the likelihood ofcalciphylaxis development (Wilmer et al. (2002) “Calciphylaxis: EmergingConcepts in Prevention, Diagnosis, and Treatment,” SEMIN. DIAL.15(3):172-86; Nigwekar et al. (2008) “Calciphylaxis from nonuremiccauses: a systematic review,” CLIN. J. AM. SOC. NEPHROL. 3(4):1139-43).In a randomized study evaluating the effect of vitamin K2supplementation on functional vitamin K deficiency in adult hemodialysispatients, patients on hemolysis (N=53) had 4.5-fold higher dp-ucMGP and8.4-fold higher uncarboxylated osteocalcin levels compared with healthyage-matched controls (N=50). PIVKA-II levels were elevated in 49hemodialysis patients. Vitamin K2 supplementation induced a dose-andtime-dependent decrease in circulating dp-ucMGP, uncarboxylatedosteocalcin, and PIVKA-II levels. Response rates in the reduction indp-ucMGP levels were 77% and 93% in the groups receiving 135 μg and 360μg of menaquinone-7, respectively (Westenfeld et al. (2012), supra).

3. Levels of Other Biomarkers for Calcification

In certain embodiments, administration of the MK-7 and/or MKH2-7increases the plasma level of osteoprotegerin or Fetuin A (e.g., by atleast 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100%) relative to the plasmaconcentration of osteoprotegerin or Fetuin A prior to administration ofthe MK-7 and/or MKH2-7. In certain embodiments, administration of theMK-7 and/or MKH2-7 increases the plasma level of osteoprotegerin orFetuin A by about 10-50%, by about 50-100%, by about 100-200%, or byabout 200-500% relative to the plasma concentration of osteoprotegerinor Fetuin A prior to administration of the MK-7 and/or MKH2-7.Osteoprotegerin and Fetuin A are inhibitors of tissue calcification.Accordingly, an increase in levels of osteoprotegerin and/or Fetuin A isindicative that administration of the MK-7 and/or MKH2-7 is successfullypreventing, slowing the progression of, arresting and/or reversingtissue calcification.

In certain embodiments, the disclosure relates to a method forincreasing the plasma level of osteoprotegerin or Fetuin A in a subjecthaving a disorder as described herein, the method includingadministering at least 2 mg/day (e.g., at least 2 mg of substantiallypure MK-7 and/or MKH2-7 per day, e.g., 10, 15, 25, 50 or 75 mg per day)MK-7 and/or MKH2-7 to the subject, wherein administration of the MK-7and/or MKH2-7 to the subject increases the plasma level ofosteoprotegerin or Fetuin A (e.g., by at least 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%) relative to the plasma concentration of osteoprotegerin or FetuinA prior to administration of the MK-7 and/or MKH2-7.

In certain embodiments of any of the above aspects, administration ofthe MK-7 and/or MKH2-7 decreases the plasma level of D-Dimers (e.g., byat least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100%) relative to the plasmaconcentration of D-Dimers prior to administration of the MK-7 and/orMKH2-7. In certain embodiments of any of the above aspects,administration of the MK-7 and/or MKH2-7 decreases the plasma level ofD-Dimers by about 10-50%, by about 50-100%, or by about 10-100% relativeto the plasma concentration of D-Dimers prior to administration of theMK-7 and/or MKH2-7. D-Dimers are indicative of the functional status ofVitamin D metabolism. Protein C and Protein S prevent generation ofthrombin and fibrin, and the lack of functional Vitamin K contributes toa loss of Protein C and Protein S, which can lead to microthrombosis viadegradation of fibrin and formation of D-Dimers. Thus presence ofD-Dimers is an indicator of sub-clinical thrombosis. Accordingly,reduction in the plasma level of D-Dimer may indicate thatadministration of the MK-7 and/or MKH2-7 is successfully restoring theGGCX carboxylation system, which can lead to preventing, slowing theprogression of, arresting and/or reversing tissue calcification.

In certain embodiments, the disclosure relates to a method fordecreasing the plasma level of D-Dimers in a subject having a disorderas described herein, the method including administering at least 2mg/day (e.g., at least 2 mg of substantially pure MK-7 and/or MKH2-7 perday, e.g., 10, 15, 25, 50 or 75 mg per day) MK-7 and/or MKH2-7 to thesubject, wherein administration of the MK-7 and/or MKH2-7 to the subjectdecreases the plasma level of D-Dimers (e.g., by at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100%) relative to the plasma concentration of D-Dimersprior to administration of the MK-7 and/or MKH2-7.

In certain embodiments of any of the above aspects, administration ofthe MK-7 and/or MKH2-7 decreases the plasma level of Highly Sensitive CReactive Protein (hs-CRP) (e.g., by at least 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%) relative to the plasma concentration of Highly Sensitive CReactive Protein (hs-CRP) prior to administration of the MK-7 and/orMKH2-7. In certain embodiments of any of the above aspects,administration of the MK-7 and/or MKH2-7 decreases the plasma level ofHighly Sensitive C Reactive Protein (hs-CRP) by about 10-50%, by about50-100%, or by about 10-100% relative to the plasma concentration ofHighly Sensitive C Reactive Protein (hs-CRP) prior to administration ofthe MK-7 and/or MKH2-7. Hs-CRP is a biomarker for ongoing inflammation.The presence of inflammation enhances soft tissue (e.g., vascular)calcification. Accordingly, reduction in the plasma level of hs-CRP canbe indicative that administration of the MK-7 and/or MKH2-7 issuccessfully preventing, slowing the progression of, arresting and/orreversing tissue calcification.

In certain embodiments, the disclosure relates to a method fordecreasing the plasma level of Highly Sensitive C Reactive Protein(hs-CRP) in a subject having a disorder as described herein, the methodincluding administering at least 2 mg/day (e.g., at least 2 mg ofsubstantially pure MK-7 and/or MKH2-7 per day, e.g., 10, 15, 25, 50 or75 mg per day) MK-7 and/or MKH2-7 to the subject, wherein administrationof the MK-7 and/or MKH2-7 to the subject decreases the plasma level ofHighly Sensitive C Reactive Protein (hs-CRP) (e.g., by at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100%) relative to the plasma concentration of HighlySensitive C Reactive Protein (hs-CRP) prior to administration of theMK-7 and/or MKH2-7.

V. Lesions

The methods and compositions described herein can be useful in slowingor arresting the progression of, arresting and/or reversing tissuelesions, for example dermal lesions or vascular lesions.

1. Dermal Lesions

The final step of the Vitamin K cycle requires the enzymatic reductionof Vitamin K 2,3-epoxide back to MK-7 by Vitamin K epoxide reductasecomplex subunit 1 (VKORC1, also referred to as VKOR (see, FIG. 1). Insome tissues, the paralog VKORC1L1 (VKORC1-Like-1) may also perform thereaction. Skin exhibits the lowest expression of VKOR-C1, consistentwith the clinical observation that Vitamin K-dependent vascularcalcification is more common in the dermis. Low skin expression ofVKOR-C1 also suggests that any condition or procedure that blocks thegeneration of Vitamin K, such as hemodialysis, predisposes skin topathologic calcification.

Thus, the methods and compositions described herein relate in part toreducing the size of a dermal lesion. In certain embodiments, thesubject has a dermal lesion, and the administration of the MK-7 and/orMKH2-7 reduces the size of the dermal lesion. In certain embodiments,administration of the MK-7 and/or MKH2-7 reduces the total surface areaof the dermal lesion by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. Incertain embodiments, administration of the MK-7 and/or MKH2-7 reducesthe total volume of the dermal lesion by at least 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 100% (i.e., the dermal lesion is eliminated).

In certain embodiments, administration of MK-7 and/or MKH2-7 reduces thetotal surface area or volume of the dermal lesion by between about 30%and about 100%, between about 30% and about 90%, between about 30% andabout 80%, between about 30% and about 70%, or between about 30% andabout 60%. In certain embodiments, administration of MK-7 and/or MKH2-7reduces the total surface area or volume of the dermal lesion by betweenabout 50% and about 100%, between about 50% and about 90%, between about50% and about 80%, between about 50% and about 70%, or between about 50%and about 60%. In certain embodiments, administration of MK-7 and/orMKH2-7 reduces the total surface area or volume of the dermal lesion bybetween about 70% and about 100%, between about 70% and about 90%,between about 70% and about 80%.

The surface area or volume of one or more skin lesions can be measuredby any method known in the art. When surface area is measured, totalsurface area can be calculated if multiple lesions are present. Volumeof a lesion can be calculated from a three-dimensional model of thelesion which is constructed from digital imaging.

In another embodiment, the effectiveness of administration of MK-7and/or MKH2-7 is determined by Von Kossa staining for calcium in adermal biopsy. This well-established stain is a validated means toexamine and quantify the amount of interstitial calcium deposition in adermal biopsy. In this approach, a skin biopsy is taken and the tissueis stained using the Von Kossa method. By binding to soft tissue andvascular deposits of phosphate, Von Kossa staining can be used todetermine whether the rate of soft tissue and vascular calcium phosphatedeposition is being slowed or reversed with administration of MK-7and/or MKH2-7. Because the calcium phosphate deposits are central topathogenesis of calciphylaxis, a reduction in Von Kossa stainingindicates a healing of the conditions that lead to calciphylaxislesions. Kits for performing the Von Kossa method are availablecommercially (see, e.g., abeam®, # ab150687).

In certain embodiments, administration of the MK-7 and/or MKH2-7 reducesthe dermal lesion by about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%,50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% (i.e., the dermallesion is eliminated) of the total surface of the lesion prior totreatment.

2. Vascular Lesions

The methods and compositions described herein relate in part to reducinga vascular lesion. In certain embodiments, the subject has a vascularlesion, and the administration of the MK-7 and/or MKH2-7 reduces thevascular lesion. Reduction in a vascular lesion can be assessed usingaortic plethysmography, which measures vascular compliance (i.e., thecapacity of a blood vessel to dilate). (See, e.g., Inuma et al. (2012)HONG KONG JOURNAL OF NEPHROLOGY 14(2):48-53.)

Reduction in a vascular lesion also can be assessed by measuring pulsewave velocity (PWV). PWV is the velocity at which the arterial pulsemoves through the circulatory system and is used as a measure ofarterial stiffness. Higher PWV corresponds to higher arterial stiffness,and therefore lower vascular compliance. Methods for measuring PWV areknown in the art and described, for example, by Pereira et al. (2015),supra. Briefly, PWV can be assessed by measuring regional PWV, such ascarotid-femoral PWV, or local PWV. Id. Methods for measuring regionalPWV are known in the art and include, for example, the use of a pulsetransducer probe, TY-360 pressure transducer, doppler unit synchronismwith electrocardiogram (ECG), pulse transducer probes,photoplethysmography, Complior® (piezoelectric pressure transducers),SphygmoCor®, Arteriograph®, and PulsePen® (tonometer and integratedelectrocardiogram unit). Id. Methods for measuring local PWV are knownin the art and include, for example, magnetic resonance imaging,ultrasound, and angiography.

In certain embodiments, administration of the MK-7 and/or MKH2-7increases vascular compliance of a blood vessel by at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% relative to vascular compliance prior totreatment. In certain embodiments, administration of the MK-7 and/orMKH2-7 increases vascular compliance of a blood vessel by about 10-50%,by about 50-100%, by about 100-200%, or by about 200-500%. In certainembodiments, administration of the MK-7 and/or MKH2-7 increases vascularcompliance of a blood vessel by at least 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%relative to vascular compliance prior to treatment. In certainembodiments, administration of the MK-7 and/or MKH2-7 increases vascularcompliance of a blood vessel by about 10-50%, by about 50-100%, by about100-200%, or by about 200-500% relative to vascular compliance prior totreatment.

VI. Kits for Use in Medical Applications

Another aspect of the invention provides a kit for treating one or moreof the disorders or conditions described herein. The kit can include: i)instructions for treating the medical disorder; and ii) one or moredosage units containing substantially pure MK-7 and/or MKH2-7.

Throughout the description, where compositions are described as having,including, or comprising specific components, or where processes andmethods are described as having, including, or comprising specificsteps, it is contemplated that, additionally, there are compositions ofthe present invention that consist essentially of, or consist of, therecited components, and that there are processes and methods accordingto the present invention that consist essentially of, or consist of, therecited processing steps.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

Throughout the description, where compositions and kits are described ashaving, including, or comprising specific components, or where processesand methods are described as having, including, or comprising specificsteps, it is contemplated that, additionally, there are compositions andkits of the present invention that consist essentially of, or consistof, the recited components, and that there are processes and methodsaccording to the present invention that consist essentially of, orconsist of, the recited processing steps.

In the application, where an element or component is said to be includedin and/or selected from a list of recited elements or components, itshould be understood that the element or component can be any one of therecited elements or components, or the element or component can beselected from a group consisting of two or more of the recited elementsor components.

Further, it should be understood that elements and/or features of acomposition or a method described herein can be combined in a variety ofways without departing from the spirit and scope of the presentinvention, whether explicit or implicit herein. For example, wherereference is made to a particular compound, that compound can be used invarious embodiments of compositions of the present invention and/or inmethods of the present invention, unless otherwise understood from thecontext. In other words, within this application, embodiments have beendescribed and depicted in a way that enables a clear and conciseapplication to be written and drawn, but it is intended and will beappreciated that embodiments may be variously combined or separatedwithout parting from the present teachings and invention(s). Forexample, it will be appreciated that all features described and depictedherein can be applicable to all aspects of the invention(s) describedand depicted herein.

The articles “a” and “an” are used in this disclosure to refer to one ormore than one (i.e., to at least one) of the grammatical object of thearticle, unless the context is inappropriate. By way of example, “anelement” means one element or more than one element.

The term “and/or” is used in this disclosure to mean either “and” or“or” unless indicated otherwise.

It should be understood that the expression “at least one of” includesindividually each of the recited objects after the expression and thevarious combinations of two or more of the recited objects unlessotherwise understood from the context and use. The expression “and/or”in connection with three or more recited objects should be understood tohave the same meaning unless otherwise understood from the context.

The use of the term “include,” “includes,” “including,” “have,” “has,”“having,” “contain,” “contains,” or “containing,” including grammaticalequivalents thereof, should be understood generally as open-ended andnon-limiting, for example, not excluding additional unrecited elementsor steps, unless otherwise specifically stated or understood from thecontext.

Where the use of the term “about” is before a quantitative value, thepresent invention also include the specific quantitative value itself,unless specifically stated otherwise. As used herein, the term “about”refers to a ±10% variation from the nominal value unless otherwiseindicated or inferred.

Where a molecular weight is provided and not an absolute value, forexample, of a polymer, then the molecular weight should be understood tobe an average molecule weight, unless otherwise stated or understoodfrom the context.

As a general matter, compositions specifying a percentage are by weightunless otherwise specified. Further, if a variable is not accompanied bya definition, then the previous definition of the variable controls.

It should be understood that the order of steps or order for performingcertain actions is immaterial so long as the present invention remainoperable. Moreover, two or more steps or actions may be conductedsimultaneously.

The use of any and all examples, or exemplary language herein, forexample, “such as” or “including,” is intended merely to illustratebetter the present invention and does not pose a limitation on the scopeof the invention unless claimed. No language in the specification shouldbe construed as indicating any non-claimed element as essential to thepractice of the present invention.

EXAMPLES

The disclosure is further illustrated by the following examples, whichare not to be construed as limiting this disclosure in scope or spiritto the specific procedures herein described. It is to be understood thatthe examples are provided to illustrate certain embodiments and that nolimitation to the scope of the disclosure is intended thereby.

Example 1—Administration of MK-7 and/or MKH2-7 in Subjects with EndStage Renal Disease (ESRD) to Reverse or Slow the Progression of TissueCalcification

This example describes the administration of MK-7 and/or MKH2-7 to asubject with ESRD and/or on stable hemodialysis. It is contemplated thatadministration of MK-7 and/or MKH2-7 will result in a change in aorticcompliance (via plethysmography), vascular calcification and certainbiomarker levels indicative of slowing the progression of, arresting, orreversing tissue calcification.

ESRD subjects on stable hemodialysis orally receive MK-7 and/or MKH2-7at 10 mg, 25 mg or 50 mg once daily for least 2 weeks, 4 weeks, 6 weeks,8 weeks, 3 months, 6 months, 1 year, or indefinitely. The dosage form isa 10 mg or 25 mg soft-gel capsule. Two 25 mg capsules are administeredonce daily to the 50 mg dosage cohort.

Coronary arterial calcium scores (CAC) are used to estimate the extentof calcification of thoracic arteries. A high CAC score is indicative ofcalcification, and treatment has the aim of arresting the long termincrease in CAC score, or reversing it, or slowing the rate of increase.Aortic plethysmography also is used to measure arterial compliance,which decreases as calcification increases. Pulse wave velocity (PWV)also is measured to assess arterial compliance. The foregoing measuresare useful in estimating the utility of treatments intended to prevent,slow the progression of, arrest or reverse vascular calcification. Thesemeasurements are used pre- and post-treatment with MK-7 and/or MKH2-7 toassess treatment value.

Further, several biomarkers are assessed to determine the efficacy ofMK-7 and/or MKH2-7 at the three dose levels. Exemplary biomarkersinclude PIVKA-II; uncarboxylated and total Matrix Gla Protein (MGP);uncarboxylated, carboxylated and total osteocalcin protein;uncarboxylated, carboxylated and total Protein C, osteoprotegerin,Fetuin A, and hs-CRP. Blood samples are obtained to measure thebiomarkers, most conveniently during patient visits for hemodialysis.

It is contemplated that administration of MK-7 and/or MKH2-7 can resultin (i) an increase in PIVKA-II, osteoprotegerin, or Fetuin A, which isindicative of slowing the progression of, arresting or reversing tissuecalcification, (ii) a decrease in uncarboxylated MGP, uncarboxylatedosteocalcin, and/or uncarboxylated Protein C, which is indicative ofslowing the progression of, arresting or reversing tissue calcification,and/or (iii) a decrease in hs-CRP, which is indicative of slowing theprogression of, arresting or reversing tissue calcification and/orreduced inflammation.

It is contemplated that, following the daily administration of 10 mg, 25mg or 50 mg MK-7 and/or MKH2-7, at least one of PIVKA-II,under-carboxylated Matrix Gla Protein (MGP), uncarboxylated osteocalcinprotein, uncarboxylated Protein C, osteoprotegerin, Fetuin A, and hs-CRPwill show a change indicative of slowing the progression of, arrestingor reversing tissue calcification.

Example 2—Administration of MK-7 and/or MKH2-7 in Subjects with StableEnd Stage Renal Disease (ESRD) Receiving Hemodialysis

This example describes the administration of MK-7 to subjects with ESRDreceiving hemodialysis, and who are at risk of developing tissuecalcification. Administration of MK-7 can result in a change in certainbiomarker levels indicative of the prevention of the development oftissue calcification, or in the arrest or slowing down of thedevelopment of tissue calcification.

A number of subjects with stable ESRD but without calciphylaxis areenrolled in the study, whereupon the subjects orally receive a 10 mgcapsule of MK-7 per day for 14 days. The levels of certain biomarkers,including uncarboxylated Matrix Gla Protein (MGP), uncarboxylatedosteocalcin, osteoprotegerin, Fetuin A and hs-CRP were assessed on day 1and on day 15 (the end of treatment), and the changes between day 1 andday 15 were calculated. Between days 1 and 15, the average reduction inuncarboxylated MGP was −21.9% and the average reduction inuncarboxylated osteocalcin was −55.5%. In addition, subjectsunexpectedly exhibited an average increase in osteoprotegerin and FetuinA and a reduction in hs-CRP. Osteoprotegerin, Fetuin A, and hs-CRP arenot Vitamin-K dependent proteins and it is believed that changes inthese proteins have not previously been demonstrated as a result of MK-7intake. The average increase in osteoprotegerin was 14.5%, the averageincrease in Fetuin A was 23.1%, and the average reduction in hs-CRP was-35.8%.

Based on the study, the administration of MK-7 can result in a decreasein uncarboxylated MGP, uncarboxylated osteocalcin, and hs-CRP, and anincrease in Fetuin A and osteoprotegerin, which can be indicative thatadministration of MK-7 prevents, or stops or slows down the progressionof tissue calcification in the subjects.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent and scientific documentsreferred to herein is incorporated by reference for all purposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

What is claimed is: 1.-18. (canceled)
 19. A method of preventing,slowing the progression of, arresting, and/or reversing tissuecalcification in a human subject in need thereof, the method comprisingadministering to the human subject at least 10 mg of substantially puremenaquinone-7 (MK-7) and/or menaquinol-7 (MKH2-7) per day thereby toprevent, slow the progression of, arrest and/or reverse tissuecalcification in the subject, whereupon the administration of the MK-7and/or the MKH2-7 increases a plasma level of Fetuin A relative to theplasma level of Fetuin A prior to administration, and at least one ofthe following: (i) increases the subject's serum T50 value relative tothe subject's serum T50 value prior to administration of the MK-7 and/orMKH2-7, (ii) increases a ratio of a carboxylated to a non-carboxylatedform of a Vitamin K-dependent protein in the subject's plasma relativeto the ratio prior to administration of the MK-7 and/or MKH2-7, (iii)increases the plasma level of osteoprotegerin relative to the plasmaconcentration of osteoprotegerin prior to administration of the MK-7and/or MKH2-7, or (iv) decreases the plasma level of D-Dimer or HighlySensitive C Reactive Protein (hs-CRP) relative to the plasmaconcentration of D-Dimer or Highly Sensitive C Reactive Protein (hs-CRP)prior to administration of the MK-7 and/or MKH2-7.
 20. The method ofclaim 19, wherein the subject has been diagnosed as pre-diabetic. 21.The method of claim 19, wherein the subject has chronic kidney disease.22. The method of claim 19, wherein the subject is undergoinghemodialysis.
 23. The method of claim 19, wherein the subject isreceiving non-warfarin-based anti-coagulant therapy.
 24. The method ofclaim 23, wherein the anti-coagulation therapy comprises an inhibitor ofFactor Xa activity or Factor IIa activity.
 25. The method of claim 19,wherein the Vitamin K-dependent protein is selected from Matrix Glaprotein, Growth Arrest Specific Gene 6 (Gas-6) protein, PIVKA-IIprotein, osteocalcin, activated Protein C, or activated Protein S. 26.The method of claim 19, comprising administering from about 10 mg toabout 100 mg of MK-7 and/or MKH2-7 to the subject per day.
 27. Themethod of claim 19, wherein the MK-7 and/or MKH2-7 is administered tothe subject for at least 2 weeks. 28.-30. (canceled)
 31. The method ofclaim 19, wherein the subject has diabetes.
 32. The method of claim 19,wherein the MK-7 and/or MKH2-7 is administered to the subject for atleast 6 weeks.
 33. The method of claim 22, wherein the MK-7 and/orMKH2-7 is administered to the subject for a period that includes theduration of hemodialysis.
 34. The method of claim 19, wherein the MK-7and/or MKH2-7 is administered orally.
 35. The method of claim 19,wherein the MK-7 and/or MKH2-7 is disposed within a tablet, caplet orcapsule.
 36. The method of claim 19, wherein the subject has previouslybeen exposed to warfarin-based anti-coagulation therapy.
 37. The methodof claim 19, wherein the subject is receiving a statin.
 38. The methodof claim 37, wherein the statin is selected from simvastatin,lovastatin, atorvastatin, pravastatin, pitavastatin, rosuvastatin, andfluvastatin.
 39. The method of claim 19, wherein the tissuecalcification is vascular calcification.
 40. The method of claim 19,wherein the tissue calcification is dermal calcification.
 41. The methodof claim 19, comprising administering 10, 25, 50, 75, or 100 mg of MK-7and/or MKH2-7 to the subject per day.